Abstract

Lignocellulosic biomass is an abundant and renewable source of aromatic compounds and carbohydrates. Microbial lignin catabolism leads to the formation of seven central aromatic intermediates that have potential markets valuated in the billions. Protocatechuate is one of the most common central intermediates and is the precursor to chemical building blocks, including cis,cis-muconic acid, used for production of bioplastics, cosmetics, food preservatives and antioxidants. Understanding the full extent of the microbial aromatic catabolic pathways is necessary to design the most efficient strains to produce valuable chemicals from protocatechuate. Aromatic catabolic pathways have been mapped out in bacteria, however, the equivalent pathways in fungi have not been as well characterized. In this study, the candidate genes NRRL3_01405, NRRL3_02586 and NRRL3_01409 encoding protocatechuate-3,4-dioxygenase, 3-carboxy-cis, cis-muconate cyclase and β-carboxymuconolactone hydrolase/decarboxylase in the Aspergillus niger protocatechuate catabolic pathway were expressed in Escherichia coli. The target proteins were purified by column chromatography and characterized by enzyme activity assays.
The products of each enzyme were characterized by time-of-flight mass spectrometry, nuclear magnetic resonance spectroscopy, and ultraviolet-visible spectroscopy. NRRL3_00837 was determined to not participate in the in vitro catabolism of protocatechuate to β-ketoadipate despite having been previously reported to participate in the in vivo protocatechuate catabolism. Mapping out the central aromatic catabolic pathways in fungi may lead to the refinement of strains engineered for lignin valorization and bioremediation.